Abstract: Amphibians are known to better tolerate and endure adverse environmental conditions such as redox imbalances conferred by reactive oxygen species (ROS), compared to mammals. Interestingly, the exact adaptation strategies and signaling mechanisms mediating these effects have not been fully elucidated. Therefore, in the present study, we probed into the molecular response of the isolated perfused Rana ridibunda heart to curcumin, in the context of mitogen-activated protein kinases (MAPKs) phosphorylation patterns and apoptotic markers occurrence. In particular, this polyphenol was found to exert a pro-oxidant effect in our model and to significantly upregulate p38-MAPK and JNKs phosphorylation (thus activation). The early apoptosis observed, substantiated by poly(ADP-ribose) polymerase (PARP) cleavage, was established to be JNKs- and ROS-mediated, while no involvement of p38-MAPK was detected. Subsequently, the pro-oxidative activity of curcumin was confirmed to mimic H(2) O(2). Furthermore, NADPH oxidase as well as Na(+) /K(+) -ATPase were found to mediate JNKs phosphorylation as well as PARP proteolytic cleavage. Curcumin exerts pleiotropic actions, both beneficial and detrimental and is currently the subject of intense scientific research. Being a low-molecular-weight antioxidant, it is intriguing to investigate curcumin's role in redox homeostasis in the amphibian heart, under conditions that apparently favor its pro-oxidative properties. Comparative studies of its multifaceted role in different species may contribute to the clarification of the signaling mechanisms it triggers and the terminal physiological response it confers. Collectively, this is to our knowledge, the first time that the signal transduction pathways stimulated by curcumin have been assessed in a non-mammalian species.

Abstract: "Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

Abstract: Nerium oleanderL. (oleander) is a very desirable ornamental, widely diffused throughout the Mediterranean region that exhibits a prolonged flowering period during the dry season. The structure of white, pink and red petals of single flowers of N. oleander was examined using light microscopy, scanning electron microscopy and atomic force microscopy. The petals consist of one-layered adaxial epidermis with conical-papillate cells, a mesophyll with loosely arranged cells and large intercellular spaces, and one-layered abaxial epidermis with cuboid cells. The adaxial and the abaxial epidermises of petals are further ornamented by wrinkled reliefs, which increase the cell surface area. Roughness of the extracellular relief varies between adaxial and abaxial surfaces of white and red petals; the opposite holds true for the pink petals of the wild plants of N. oleander. Roughness was linearly correlated with vertical distance, and surface distance was linearly correlated with horizontal distance of the reliefs. Micromophology of petals of N. oleander revealed sub-micron features that may be particularly important for the performance of the nectarless flowers under ambient conditions, affecting energy exchange with the surrounding environment and adhesive properties of pink flowers during a seven-day flower lifespan, and white and red flowers during a five-day lifespan

Abstract: The objective of this work was to compare and contrast the patterns of alien plant invasions in the world’s five mediterranean-climate regions (MCRs). We expected landscape age and disturbance history to have bearing on levels of invasion. We assembled a database on naturalized alien plant taxa occurring in natural and semi-natural terrestrial habitats of all five regions (specifically Spain, Italy, Greece and Cyprus from the Mediterranean Basin, California, central Chile, the Cape Region of South Africa and Southwestern - SW Australia). We used multivariate (hierarchical clustering and NMDS ordination) trait and habitat analysis to compare characteristics of regions, taxa and habitats across the Mediterranean biome. Our database included 1627 naturalized species with an overall low taxonomic similarity among the five MCRs. Herbaceous perennials were the most frequent taxa, with SW Australia exhibiting both the highest numbers of naturalized species and the highest taxonomic similarity (homogenization) among habitats, and the Mediterranean Basin the lowest. Low stress and highly disturbed habitats had the highest frequency of invasion and homogenization in all regions, and high natural stress habitats the lowest, while taxonomic similarity was higher among different habitats in each region than among regions. Our analysis is the first to describe patterns of species characteristics and habitat vulnerability for a single biome. We have shown that a broad niche (i.e. more than one habitat) is typical of naturalized plant species, regardless of their geographical area of origin, leading to potential for high homogenization within each region. Habitats of the Mediterranean Basin are apparently the most resistant to plant invasion, possibly because their landscapes are generally of relatively recent origin, but with a more gradual exposure to human intervention over a longer period.

Abstract: G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit's behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors.

Abstract: The development of a novel approach for the prediction of antiestrogenic activity is described, bringing up to date a previous pharmacophore study. Software Phase has been used to derive a 3D-QSAR model based, as alignment rule, on a pharmacophore built on three compounds highly active against MCF-7 cell line. Five features comprised the pharmacophore: two hydrogen-bond acceptors, one hydrogen-bond donor, and two aromatic rings. The sequential 3D-QSAR yielded a test set q(2) equal to 0.73 and proved to be predictive with respect to an external test set of 21 compounds (r(2) = 0.69). The model was used to detect new MCF-7 inhibitors through 3D-database searching and identified fourteen compounds that were subsequently tested in vitro against the MCF-7 human breast adenocarcinoma cell line. Eleven out of the fourteen compounds exhibited inhibitory activity with IC50 values ranging between 30 and 186 μM. The results of the study confirmed the fundamental validity of the chosen approach as a hit discovery tool.

Abstract: The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer's disease (AD). It has become increasingly evident that AβPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AβPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AβPP-) or increased expression of AβPP695 by transfection (SH-SY5Y/AβPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AβPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AβPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AβPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AβPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AβPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AβPP in calcium homeostasis and in AD.

Abstract: Transcription of the mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis has been studied by RT-PCR and RNA circularization. This species has an egg-transmitted (F) and a sperm-transmitted (M) mitochondrial genome, in accordance with the doubly uniparental inheritance (DUI) pattern of mtDNA transmission. The primary transcript is cleaved into ten transcripts, eight of which are monocistronic, one is tricistronic and one is most likely, but not certainly, bicistronic. The start/end points of these transcripts have been determined. In the majority of cases cleavage is mediated according to the "tRNA punctuation" model. However, we have identified four cases of cleavage that do not coincide with the presence of a tRNA. In these cases transcription starts immediately or only a few bases from the end point of the preceding gene and cleavage is, most likely, mediated by a stem-loop structure formed at the start point of the gene. The identification of a tricistronic transcript is a novel finding for metazoan mtDNA. We propose that its evolution has been facilitated by the fact that all coding genes are transcribed from the same DNA strand and that co-transcription is sustained by selection emanating from the fact that proteins derived from all three co-transcribed genes participate in the formation of the same oxidative phosphorylation complex.

Abstract: The purpose of this study is to assess sexual dimorphism in the palate and base of adult crania using three-dimensional geometric morphometric methods. The study sample consisted of 176 crania of known sex (94 males, 82 females) belonging to individuals who lived during the 20th century in Greece. The three-dimensional co-ordinates of 30 ectocranial landmarks were digitized using a MicroScribe 3DX contact digitizer. Generalized Procrustes Analysis (GPA) was used to obtain size and shape variables for statistical analysis. Three discriminant function analyses were carried out: (1) using PC scores from Procrustes shape space, (2) centroid size alone, and (3) PC scores of GPA residuals which includes lnCS for analysis in Procrustes form space. Results indicate that there are shape differences between sexes. In males, the palate is deepest and more elongated; the cranial base is shortened. Sex-specific shape differences for the cross-validated data give better classification results in the cranial base (77.2%) compared with the palate (68.9%). Size alone yielded better results for cranial base (82%) in opposition to palate (63.1%). As anticipated, the classification accuracy improves when both size and shape are combined (90.4% for cranial base, and 74.8% for palate).

Abstract: The dorsiventral, amphistomatic, hairy leaf of Pelargonium odoratissimum, although exposed to the stressing conditions of the Mediterranean climate, is of light structure, compared to the leaf of most Mediterranean xerophytes, lucking the variety of the anatomical features considered so far as advantages for the Mediterranean plant life. On the other hand, it has a well equipped and intensely functioning secretory apparatus. Light, scanning and transmission electron microscopy were employed to investigate the leaf structure while histochemical reactions traced the nature of the secreted metabolites. The numerous trichomes of the leaf are very active in producing phenolics, terpenes and flavonoids all mixed in a fragrant essential oil accumulated in an ovoid chamber at the top of each secreting hair. In-vitro cell cultures are suggested for the production of novel compounds from low-cost precursors.

Abstract: The dorsiventral, amphistomatic, hairy, summer and winter leaves of Cistus creticus, present a series of anatomical features indicating a very successful adaptation to the Mediterranean plant life. The secretory apparatus of this species seems rather “innovative” since the secreted resinous material is not preserved in a cutinous wrap, as commonly happens during excretion in xerophytes, but is directly spread all over the leaf surface and the protective trichomes. Summer and winter leaves present interesting structural differences. All mesophyll cells accumulate large amounts of secondary metabolites, mainly of phenolic nature. In-vitro cell cultures are suggested for the production of novel compounds from low-cost precursors.

Abstract: The anatomy of the dorsiventral, amphistomatic leaf of Papaver rhoeas is investigated. It is of a simple structure possessing large epidermal cells with their vacuole impregnated with glycoproteins. These compounds vary in texture and they usually form a dense, hard core in the middle of the vacuole. Slender rays of cisternae radiate from the dense central core to the cell wall. Thin-walled palisade cells present a unique lobed shape surely creating larger surfaces for gas exchange. The leaf has rarely observed accommodation for the stomata, especially on the adaxial epidermis, offering an effective protection while the secreting activity of the mesophyll cells is remarkable. Phenolic compounds, condensed tannins, alkaloids, terpene containing steroids and sesquiterpenes are produced. Finally it seems that P. rhoeas although being a therophyte, has also adopted some structural features common in many mediterranean phanerophytes.

Abstract: In the past few decades there is an increasing trend of both fire activity and area burned in many regions of the world. Moreover, there is a worldwide concern regarding the increasing presence of crown fires in forest types that were historically prone to surface fires. Among the recently affected mountainous forest ecosystems are those of Pinus nigra, an ecologically and economically important species that is widely distributed around the Mediterranean Basin. Mount Taygetos, a mountainous landscape in Peloponnese, Greece, that was severely burned in 2007, was selected to carry out the first landscape-scale fire history reconstruction in P. nigra of the eastern Mediterranean. The aims of the study were to investigate whether fire-regime attributes can be reconstructed from fire-scarred trees and also to examine the consistency of fire occurrence and spatial extent through time within the area selected. Partial cross-sections were sampled within the perimeters of the more recent known large fires in the region, those of 2007 and 1998. The overall mean fire interval between 1845 and 2007 was 4.9 years, while for the larger fires this time window was 16.2 years. Even at the individual-sample scale, with the sample mean fire interval equalling 29.5 years, the fire frequency still falls within the range of the ‘‘predictable stand-thinning fire’’ regime. The majority of fire scars recorded were dated to the warm and dry season of summer to fall. During the last 165 years of fire reconstruction, neither fire frequency nor percentage of trees scarred by fires varied significantly. Nevertheless, the size of the area burned as well as the type of fire seem to have changed, with the 2007 event being the most extended crown fire encountered so far. Our study has provided additional evidence that P. nigra is indeed a fire-resistant tree species provided that it is exposed to surface fires, even if they are recurrently occurring. Shifts from this pattern may lead to local extirpation of the species, as in the case of severe and extended crown fires.

Abstract: Gender seems to interfere with the cardioprotective effect of ischemic preconditioning (PreC) and postconditioning (PostC); PreC-conferred protection is weaker or lost in female animals after ovariectomy (Ov), while the role of PostC is still in dispute. We sought to investigate the effect of PostC in female rabbits, its interaction with Ov, and the potential implicated intracellular pathways. Intact or Ov adult female rabbits (n = 46) were subjected to 30 min ischemia and reperfusion with PostC (PostC or OvPostC), which consisted of six cycles of 30-s ischemia/30-s reperfusion at the end of ischemia, or without PostC (Fem or OvFem). Infarct size (I) and area at risk (R) were determined by TTC staining and fluorescent particles, respectively, after 3-h reperfusion in 30 out of 46 animals. Plasma levels of estradiol and nitrite/nitrate (NO x ) were evaluated. ERKs, p38-MAPK, and Akt assessment was performed in excised hearts 1-min after starting the final reperfusion period in the remaining 16 animals. Infarct size was significantly reduced only in OvPostC group (I/R ratio, 25.3 ± 2.7, vs 48.1 ± 2.0, 43.6 ± 4.2 and 55.1 ± 5.6 % in Fem, OvFem, and PostC groups, p < 0.05). In ovariectomized rabbits, plasma estradiol and NO x levels were lower than in the normal ones. Akt phosphorylation in ischemic regions was significantly higher in OvPostC group, whereas ERK1/2 and p38-MAPK activation was observed in all ovariectomized animals irrespective of PostC. PostC is not effective in female rabbits, but the protection is reinstated after Ov potentially via the RISK pathway.

Diallinas G. (2013) Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter. Biochimie 95:1755-64

Abstract: In Aspergillus nidulans UapA is a H(+)-driven transporter specific for xanthine, uric acid and several analogues. Here, genetic and physiological evidence is provided showing that allopurinol is a high-affinity, low-capacity, substrate for UapA. Surprisingly however, transport kinetic measurements showed that, uniquely among all recognized UapA substrates, allopurinol is transported by apparent facilitated diffusion and exhibits a paradoxical effect on the transport of physiological substrates. Specifically, excess xanthine or other UapA substrates inhibit allopurinol uptake, as expected, but the presence of excess Allopurinol results in a concentration-dependent enhancement of xanthine binding and transport. Flexible docking approaches failed to detect Allopurinol binding in the major UapA substrate binding site, which was recently identified by mutational analysis and substrate docking using all other UapA substrates. These results and genetic evidence suggest that the allopurinol translocation pathway is distinct from, but probably overlapping with, that of physiological UapA substrates. Furthermore, although the stimulating effect of allopurinol on xanthine transport could, in principle, be rationalized by a cryptic allopurinol-specific allosteric site, evidence was obtained supporting that accelerated influx of xanthine is triggered through exchange with cytoplasmically accumulated allopurinol. Our results are in line with recently accumulating evidence revealing atypical and complex mechanisms underlying transport systems.

Abstract: In the present study, we revised Albanian Agathylla, combining the traditional shell morphological approach with a phylogenetic reconstruction based on three mitochondrial DNA markers (COI, COII and 16S rRNA). From the faunistic perspective, Albania was an almost completely unexplored country until the end of the 1990s. Following recent intense field sampling activity in Albania, the number of known Agathylla populations has increased, providing an opportunity to investigate how additional material can contribute to the resolution of the taxonomy of the Albanian Agathylla species. Newly discovered populations indicate greater morphological variability than previously recorded and highlight the presence of various other character state combinations. Based on these findings, we argue that only two Agathyllataxa occur in the region, namely A. neutra and A. biloba. Due to apparent morphological differences, they are still treated as distinct species, but high intraspecific morphological variability makes it unjustifiable to delimit subspecies. The molecular phylogeny indicates a close relationship between A. neutra and A. biloba, but could neither confirm nor refute their reciprocal monophyly, so their possible conspecificity requires further evaluation.

Abstract: Chorion proteins of Lepidoptera have a tripartite structure, which consists of a central domain and two, more variable, flanking arms. The central domain is highly conserved and it is used for the classification of chorion proteins into two major classes, A and B. Annotated and unreviewed Lepidopteran chorion protein sequences are available in various databases. A database, named LepChorionDB, was constructed by searching 5 different protein databases using class A and B central domain-specific profile Hidden Markov Models (pHMMs), developed in this work. A total of 413 Lepidopteran chorion proteins from 9 moths and 1 butterfly species were retrieved. These data were enriched and organised in order to populate LepChorionDB, the first relational database, available on the web, containing Lepidopteran chorion proteins grouped in A and B classes. LepChorionDB may provide insights in future functional and evolutionary studies of Lepidopteran chorion proteins and thus, it will be a useful tool for the Lepidopteran scientific community and Lepidopteran genome annotators, since it also provides access to the two pHMMs developed in this work, which may be used to discriminate A and B class chorion proteins. LepChorionDB is freely available at http://bioinformatics.biol.uoa.gr/LepChorionDB

Abstract:Background and Aims: The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation.Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1_3,1_4)-β-D-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Methods: Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Results: Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. Conclusions: The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

Abstract: Molecular Recognition Features (MoRFs) are defined as short, intrinsically disordered regions in proteins that undergo disorder-to-order transition upon binding to their partners. As their name suggests, they are implicated in molecular recognition, which serves as the initial step for protein-protein interactions. Membrane proteins constitute approximately 30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. The aim of the current study was to identify and analyze MoRFs in membrane proteins. Two datasets of MoRFs, transmembrane and peripheral membrane protein MoRFs, were constructed from the Protein Data Bank, and sequence, structural and functional analysis was performed. Characterization of our datasets revealed their unique compositional biases and membrane protein MoRFs were categorized depending on their secondary structure after the interaction with their partners. Moreover, the position of transmembrane protein MoRFs in relation with the protein's topology was determined. Further studies were focused on functional analyses of MoRF-containing proteins and MoRFs' partners, associating them with protein binding, regulation and cell signaling, indicating half of them as putative hubs in protein-protein interaction networks. In conclusion, we provide insights into the disorder-based protein-protein interactions involving membrane proteins.

Abstract: Reactive oxygen species (ROS) imbalance is a stressful condition for plant cells accompanied by dramatic changes in tubulin cytoskeleton. Here, evidence is provided that alterations in ROS levels directly interfere with the phosphorylation state of a p38-like MAPK in the angiosperms Triticum turgidum and Arabidopsis thaliana. Both oxidative stress generators and chemicals inducing ROS scavenging or decreasing ROS production resulted in the accumulation of a phospho-p46 protein similar to p38-MAPK. Importantly, the rhd2 A. thaliana mutants exhibited a remarkable increase in levels of phospho-p46.The presence of the p38-MAPK inhibitor SB203580 attenuated the response to ROS disturbance, prevented microtubule disappearance and resulted in a dramatic decrease in the number of atypical tubulin polymers. Moreover, in roots treated simultaneously with substances inducing ROS overproduction and others resulting in low ROS levels, phospho-p46 levels and the organization of tubulin cytoskeleton were similar to controls. Collectively, our experimental data suggest, for the first time in plants, that p46 functions as a putative sensor of redox state, the activation of which initiates downstream signaling events leading to microtubule disruption and subsequent assembly of atypical tubulin polymers. Thus, p46 seems to participate in perception of ROS homeostasis disturbance as well as in cellular responses to redox imbalance.

Abstract: The scorpion genus Euscorpius has been insufficiently studied on the Aegean Islands of Greece. We describe a new species from the Western Cyclades, E. kritscheri sp. n., from Tinos Island. The species-level status of this form is confirmed by a phylogenetic study based on multiple DNA markers. In addition, a population from nearby Andros Island is characterized as E. cf. kritscheri, which could be potentially a separate species.

Abstract: In Greece, scorpion genus Euscorpius has been insufficiently studied. Taxonomy of several species and subspecies has been inconsistent and confusing. We provide new morphological data and redescriptions of type specimens of three “old” taxa, described from Greece and formerly listed under a “catch-all” taxon Euscorpius carpathicus. We elevate to, or confirm at species status: Euscorpius scaber Birula, 1900 (type locality: Mt. Athos), E. candiota Birula, 1903 (type locality: Heraklion, Crete), and E. ossae Di Caporiacco, 1950, stat.n. (type locality: Mt. Ossa, Thessaly). Species-level separation of these taxa is also confirmed by multiple species delimitation methods implemented on the phylogenetic data generated using four different DNA markers.

Abstract: Molecular recognition features (MoRFs) are small, intrinsically disordered regions in proteins that undergo a disorder-to-order transition on binding to their partners. MoRFs are involved in protein-protein interactions and may function as the initial step in molecular recognition. The aim of this work was to collect, organize and store all membrane proteins that contain MoRFs. Membrane proteins constitute ∼30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. MoRFs were classified according to their secondary structure, after interacting with their partners. We identified MoRFs in transmembrane and peripheral membrane proteins. The position of transmembrane protein MoRFs was determined in relation to a protein's topology. All information was stored in a publicly available mySQL database with a user-friendly web interface. A Jmol applet is integrated for visualization of the structures. mpMoRFsDB provides valuable information related to disorder-based protein-protein interactions in membrane proteins.

Abstract: Calcitonin is a 32-residue polypeptide hormone, which takes part in calcium metabolism in bones. It may form amyloid fibrils. Amyloid fibrils are related with serious diseases known as amyloidoses. The amyloid form of calcitonin takes part in medullary thyroid carcinoma. A novel hexapeptide ((6)TCMLGT(11)) of human calcitonin was predicted as a possible 'aggregation-prone' peptide, which may play a role in amyloid formation. We investigated experimentally the ability of an analog of this hexapeptide (cysteine replaced by alanine, TAMLGT) to form amyloid fibrils utilizing TEM, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized light microscopy. This peptide self-assembles into amyloid-like fibrils and fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites.

Abstract: We investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. A single arrestin-like protein, ArtA, is essential for HulA(Rsp) (5) –dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Mutational analysis showed that residues 545-563 of the UapA C-terminal region are required for efficient UapA endocytosis, whereas the N-terminal region (residues 2-123) and both PPxY motives are essential for ArtA function. We further show that ArtA undergoes HulA-dependent ubiquitination at residue Lys-343 and that this modification is critical for UapA ubiquitination and endocytosis. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or l-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestin-like proteins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestin-like proteins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.

Abstract: Nanoparticles prepared from biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and activate immune responses. In this study, fluorescently labelled bovine serum albumin (BSA-FITC)-loaded PLGA nanoparticles, of an average size ∼300 nm were prepared and examined for their ability to be taken up by bone marrow-derived dendritic cells (BM-DCs) in vitro and thus to promote their maturation and activation. The synthesized nanoparticles did not exhibit any cytotoxic or hemolytic effect and were taken up by BM-DCs efficiently, in a time and dose dependent manner. The localization of BSA-FITC loaded PLGA nanoparticles both in the acidophilic cellular compartments and the cytoplasm resulted in the maturation of BM-DCs expressing higher levels of costimulatory and MHC class II molecules in comparison to empy PLGA nanoparticles. However, the absence of IL-12 or IL-10 production indicates partial activation of BM-DCs suggesting the necessity of an adjuvant addition in order to facilitate DCs functionalization.

Abstract: Historical fire records and meteorological observations, spanning more than 1 century (1894–2010), were gathered and assembled in a database, to provide long-term fire–weather associations. We investigated the relationships between forest fire activity and meteorological parameters and sought to find temporal patterns and trends in these historical records and to identify any linkages between meteorological parameters and fire occurrence in the eastern Mediterranean region. Trend analysis of the time series revealed a statistically significant increase in the number of fires and air temperature, particularly after the mid-1970s. Fire occurrence, expressed as the annual number of fires and total burnt area, was strongly correlated with the mean maximum and the absolute maximum air temperature which, in turn, was related to the occurrence of summer heat waves. Total burnt area was also strongly negatively correlated with fire-season precipitation, and positively correlated with 2-year-lagged annual and summer precipitation, underlying the effect of precipitation in controlling fuel production and moisture. These findings support the argument that although annually lagged precipitation totals may have a marginal effect on fire risk by influencing biomass production and accumulation, the lag0 weather parameters are the main drivers of fire spread by directly controlling fuel moisture.

Abstract: The type of branching is a morphological character of great significance, separating taxa within the true-branched cyanobacteria. In this paper, true-branched morphospecies of cyanobacteria belonging to genus Iphinoe Lamprinou et Pantazidou, from different and geographically isolated caves of Greece, were investigated by coupling classical approach, i.e. microscopy analysis (LM, SEM, TEM, CLSM) and molecular analysis (16S rDNA gene diversity). The morphological study revealed interspecific differences which could be attributed to adaptations to local conditions: Iphinoe cf. spelaeobios from the cave Diktaion Andron’(Crete island) exhibit different type of branching (Y- and T-type) and smaller size compared to the type species Iphinoespelaeobios (T- and V-type of branching) from the caves ‘Kastria’and Koutouki’(Peloponnese, Attica). Despite their phenotypic differences and geographical isolation molecular analysis showed identical 16S rRNA sequences

Abstract: Specimens of cyanobacteria were collected from three geographically distant caves in Greece, and an interesting species of the Oscillatoriales was isolated and analyzed. Morphological characters were analyzed from fresh and cultured material using LM, SEM and TEM; moreover, molecular analysis based on the 16 S rRNA sequence was applied. This cyanobacterium bears a characteristic blackish and stratified, mucilaginous sheath and it is characterized by entangled filaments, facultative presence of false branching, presence of necridia and peripheral arrangement of thylacoids. The autapomorphic blackish mucilaginous sheath and the pseudobranching filaments are unique morphological characters within the genus Phormidium, according to classical taxonomy. The 16 S rRNA sequence showed relatedness to Phormidiumpriestleyi ANT L52.6 with 97-98 % similarity, but without morphological support. Both phenotypic and genetic traits supported the designation of a new species, under the name Phormidiummelanochroun Lamprinou & Pantazidou sp. nov.

Abstract: Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.

Abstract: Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as 'aggregation-prone' by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted 'aggregation-prone' parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization.

Abstract: Isolated atrial amyloidosis (IAA) is a common localized form of amyloid deposition within the atria of the aging heart. The main constituents of amyloid fibrils are atrial natriuretic peptide (ANP) and the N-terminal part of its precursor form (NT-proANP). An 'aggregation-prone' heptapeptide ((114)KLRALLT(120)) was located within the NT-proANP sequence. This peptide self-assembles into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies reveal. Consequently, remedies/drugs designed to inhibit the aggregation tendency of this 'aggregation-prone' segment of NT-proANP may assist in prevention/treatment of IAA, congestive heart failure (CHF) or atrial fibrillation (AF).

Abstract: Grazing of goats on Mediterranean islets is a common practice. Its consequences on plant communities are well documented, although not on vertebrates. We aim to shed light on the effect of livestock arming on lizards by investigating five populations of the insular lizard, Podarcis gaigeae, differing in the duration and intensity of grazing. Data on grazing regime, invertebrate abundance, tick prevalence, infestation levels, gull nests and lizard densities were collected during a period of 6 consecutive years. Grazing had a negative impact on insect populations,thus decreasing food availability for lizards. Tick prevalence and infestation levels were higher in places of continuous grazing. Goat activity disturbed gulls, which avoid nesting, so depriving the islets of marine subsidies. As a consequence of all these factors, lizard densities were higher in ungrazed and lower in grazed biotopes. Grazing effects were more severe on islets communities than on the mainisland populations. Our data imply that management action should be taken to conserve the highly diverse islet populations.

Abstract: Pulmonate snails have remarkably high levels of mtDNA polymorphism within species and divergence between species, making them an interesting group for the study of mutation and selection on mitochondrial genomes. The availability of sequence data from most major lineages - collected largely for studies of phylogeography - provides an opportunity to perform several tests of selection that may provide general insights into the evolutionary forces that have produced this unusual pattern. Several protein coding mtDNA datasets of pulmonates were analyzed towards this direction. Two different methods for the detection of positive selection were used, one based on phylogeny, and the other on the McDonald-Kreitman test. The cyto-nuclear coevolution hypothesis, often implicated to account for the high levels of mtDNA divergence of some organisms, was also addressed by assessing the divergence pattern exhibited by a nuclear gene. The McDonald-Kreitman test indicated multiple signs of positive selection in the mtDNA genes, but was significantly biased when sequence divergence was high. The phylogenetic method identified five mtDNA datasets as affected by positive selection. In the nuclear gene, the McDonald-Kreitman test provided no significant results, whereas the phylogenetic method identified positive selection as likely present. Overall, our findings indicate that: 1) slim support for the cyto-nuclear coevolution hypothesis is present, 2) the elevated rates of mtDNA polymorphims and divergence in pulmonates do not appear to be due to pervasive positive selection, 3) more stringent tests show that spurious positive selection is uncovered when distant taxa are compared and 4) there are significant examples of positive selection acting in some cases, so it appears that mtDNA evolution in pulmonates can escape from strict deleterious evolution suggested by the Muller's ratchet effect.

Abstract: Euscorpius avcii Tropea et al., 2012 has been recently described from Dilek Peninsula in western Anatolia (Turkey, Aydın Province). The population from Samos Island in eastern part of the Aegean Sea is found to match closely the Anatolian E. avcii, making it a new, rare species for the Greek fauna, confirmed by two DNA markers as well as morphology. Samos also shares with western Anatolia two other local recently described scorpion species, Iurus kinzelbachi and Neocalchas gruberi (family Iuridae).

Abstract: The objectives of modeling in this work were (a) the integration of two existing numerical models in order to connect external exposure to nanoparticles (NPs) with internal dose through inhalation, and (b) to use computational fluid-particle dynamics (CFPD) to analyze the behavior of NPs in the respiratory and the cardiovascular system. Regarding the first objective, a lung transport and deposition model was combined with a lung clearance/retention model to estimate NPs dose in the different regions of the human respiratory tract and some adjacent tissues. On the other hand, CFPD was used to estimate particle transport and deposition of particles in a physiologically based bifurcation created by the third and fourth lung generations (respiratory system), as well as to predict the fate of super-paramagnetic particles suspended in a liquid under the influence of an external magnetic field (cardiovascular system). All the above studies showed that, with proper refinement, the developed computational models and methodologies may serve as an alternative testing strategy, replacing transport/deposition experiments that are expensive both in time and resources and contribute to risk assessment.

Abstract: Isimeropesemele n. gen. and n. sp., a valvatiform hydrobiid from southern continental Greece, is described based on morphological and molecular data. Isimerope is distinguished from other European and circum-Mediterranean valvatiform hydrobiid genera by a unique combination of morphological characters, including distinctive male and female genitalia. Isimerope is differentiated from morphologically similar Graecoarganiella, which is also endemic to Greece, by a 10.15% mean COI sequence divergence. Isimerope semele is composed of three small populations living in disturbed habitats, including springs and a river.

Abstract: Body size shapes the overall biology of organisms. We assessed the impact of size on temperature regulation in populations of normal-sized and large-bodied insular Mediterranean lizards (Podarcis gaigeae, Lacertidae). We hypothesized that large lizards would achieve higher body temperatures and thermoregulate more effectively than their smaller kin. Large- and small-bodied lizards share the same thermoregulation pattern, achieving similar body temperatures in the field. Large lizards, however, prefer higher set-point temperatures. Lizards in both populations thermoregulate effectively, but large lizards thermoregulated less effectively than normal-sized lizards. The particular conditions at the islet that harbors the large-bodied population (harsh intraspecific competition) seem to account for this pattern.

Abstract: The overall biology of ectotherms is strongly affected by the thermal quality of the environment. The particular conditions prevailing on islands have a strong effect on numerous features of animal life. In this study we compared mainland and island populations of the lizard Lacerta trilineata and hypothesized that insularity would affect the thermoregulatory strategy. Continental habitats were of lower thermal quality, experiencing more intense fluctuations and had higher values of operative temperatures. Nevertheless mainland lizards selected for higher body temperatures in the lab and showed more effective thermoregulation during summer than their island peers. Lizards achieved similar body temperatures in the field in both types of habitat, underlining the importance of predation as a potential factor to mainland lizards that failed to reach their higher thermal preferences. Both island and mainland populations of L. trilineata have been adapted to their thermal environment, supporting the labile view on the evolution of thermal physiology for this species.

Abstract: Mountains of the northern Mediterranean basin face two major threats under global change. Aridity and available fuel are both expected to increase because of climatic and land-use changes, increasing fire danger. There may already be signs of such effects in the case of the Pinus nigra and Abies cephalonica forests on Mt. Taygetos (southern Greece). We reconstructed climate (mid- to late-fire-season drought) using tree-rings for the last 150 years and compared it with the mountain’s fire history reconstructed from P. nigra fire scars. Seven, out of the ten, large fires Mt. Taygetos experienced were associated with below-normal precipitation (P) or above-normal maximum temperature (Tmax). The largest fires occurred in late summer of 1879, 1944, 1998, and 2007. However, only the recent fires (1998 and 2007) had both low P and high Tmax, also confirmed from long-term meteorological data. The synergy between climate and fuel availability may explain the very high intensity of 1998 and 2007 fires that burned mostly as stand-replacing crown fires. The other two large fire events (1879 and 1944) most likely occurred under reduced availability in burning fuel and were related to above-normal Tmax. Our findings are among the first based on long-term and site-specific empirical data to support the prediction that Mediterranean mountainous areas will face a very large threat from wildfires in the twenty-first century, if socioeconomic changes leading to land abandonment and thus burning fuel accumulation are combined with the drought intensification projected for the region under global warming.

Abstract: In fungi, transcriptional activation of genes involved in NO3(-) assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an "autoregulation control" model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show "pseudo-constitutivity" due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO3 accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed "pseudo-constitutive".

Abstract: For the Mediterranean Sea, and indeed most of the world’s oceans, the biodiversity and biogeography of eukaryotic pathogens infecting marine macroalgae remains poorly known, yet their ecological impact is probably significant. Based on 2 sampling campaigns on the Greek island of Lesvos in 2009 and 1 in northern Greece in 2012, this study provides first records of 3 intracellular eukaryotic pathogens infecting filamentous brown algae at these locations: Eurychasma dicksonii, Anisolpidium sphacellarum, and A. ectocarpii. Field and microscopic observations of the 3 pathogens are complemented by the first E. dicksonii large subunit ribosomal RNA (LSU rRNA) gene sequence analyses of isolates from Lesvos and other parts of the world. The latter highlights the monophyly of E. dicksonii worldwide and confirms the basal position of this pathogen within the oomycete lineage (Peronosporomycotina). The results of this study strongly support the notion that the geographic distribution of the relatively few eukaryotic seaweed pathogens is probably much larger than previously thought and that many of the world’s marine bioregions remain seriously undersampled and understudied in this respect.

Abstract: A new scorpion species, Euscorpius (Euscorpius) erymanthius sp. n., is described from Peloponnese, Greece (Erymanthos Mts.), based on genetic and morphological evidence. It is characterized by small size, light brown to reddish color, and a standard trichobothrial pattern (Pv = 8–9, et = 7–6, em = 4 and eb = 4). In a phylogeny based on multiple DNA markers, the new species groups close with E. corcyraeus Tropea et Rossi, 2012 from Corfu (Kerkyra) Island.

Abstract: Marine benthic macroalgae populations have been qualitatively and quantitatively studied for the first time from the eastern coasts of Andros Island (Aegean Sea, Greece). Samples were collected from rocky shores at the capital city (Andros Village) and nearby sites. A total of 113 taxa were identified, comprising of 24 Chlorophyta, 23 Ochrophyta and 66 Rhodophyta. A bushy phytocommunity dominated by geniculate red algae was observed at sites at a distance from ‘Andros Village’ whilst at the latter the alien macroalgae Codiumfragile subsp. fragile and Asparagopsistaxiformis prevailed. These taxa exhibited invasive behavior locally displaying extremely high abundance. The canopy forming Cystoseira species were present at all studied sites but only in low abundances.

Abstract: An updated checklist of the brown seaweeds (Phaeophyceae) of Greece is provided, based on both literature records and new collections. The total number of species and infraspecific taxa currently accepted is 107. The occurrence of each taxon in the North Aegean, South Aegean and Ionian Seas is given. In addition, 17 taxa pending confirmation of their presence, 11 excludenda and 8 inquirenda are briefly discussed.

Abstract: Oomycete diseases in seaweeds are probably widespread and of significant ecological and economic impact, but overall still poorly understood. This study investigates the organisation of the cytoskeleton during infection of three brown algal species, Pylaiella littoralis, Ectocarpus siliculosus, and Ectocarpus crouaniorum, by the basal marine oomycete Eurychasma dicksonii. Immunofluorescence staining of tubulin revealed how the development of this intracellular biotrophic pathogen impacts on microtubule (MT) organisation of its algal host. The host MT cytoskeleton remains normal and organised by the centrosome until very late stages of the infection. Additionally, the organisation of the parasite’s cytoskeleton was examined. During mitosis of the E. dicksonii nucleus the MT focal point (microtubule organisation centre, MTOC, putative centrosome) duplicates and each daughter MTOC migrates to opposite poles of the nucleus. This similarity in MT organisation between the host and pathogen reflects the relatively close phylogenetic relationship between oomycetes and brown algae. Moreover, actin labelling with rhodamine-phalloidin in E. dicksonii revealed typical images of actin dots connected by fine actin filament bundles in the cortical cytoplasm. The functional and phylogenetic implications of our observations are discussed.

Tsirigoti, A., Küpper F.C., Gachon, C.M.M., Katsaros, C. (2013) Filamentous brown algae infected by the marine, holocarpic oomycete Eurychasma dicksonii: first results on the organization and the role of cytoskeleton in both host and parasite. Plant Sign. Behav. doi.org/10.4161 /psb.26367

Abstract: The important role of the cytoskeletal scaffold is increasingly recognized in host-pathogen interactions. The cytoskeleton potentially functions as a weapon for both the plants defending themselves against fungal or oomycete parasites, and for the pathogens trying to overcome the resisting barrier of the plants. This concept, however, had not been investigated in marine algae so far. We are opening this scientific chapter with our study on the functional implications of the cytoskeleton in 3 filamentous brown algal species infected by the marine oomycete Eurychasma dicksonii. Our observations suggest that the cytoskeleton is involved in host defense responses and in fundamental developmental stages of E. dicksonii in its algal host.

Abstract: The purpose of this work was to construct a consensus prediction algorithm of 'aggregation-prone' peptides in globular proteins, combining existing tools. This allows comparison of the different algorithms and the production of more objective and accurate results. Eleven (11) individual methods are combined and produce AMYLPRED2, a publicly, freely available web tool to academic users (http://biophysics.biol.uoa.gr/AMYLPRED2), for the consensus prediction of amyloidogenic determinants/'aggregation-prone' peptides in proteins, from sequence alone. The performance of AMYLPRED2 indicates that it functions better than individual aggregation-prediction algorithms, as perhaps expected. AMYLPRED2 is a useful tool for identifying amyloid-forming regions in proteins that are associated with several conformational diseases, called amyloidoses, such as Altzheimer's, Parkinson's, prion diseases and type II diabetes. It may also be useful for understanding the properties of protein folding and misfolding and for helping to the control of protein aggregation/solubility in biotechnology (recombinant proteins forming bacterial inclusion bodies) and biotherapeutics (monoclonal antibodies and biopharmaceutical proteins).